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Name: Downloaded from www.pharmacy123.blogfa.com کروماتوگرافی CHROMATOGRAPHY Downloaded from www.pharmacy123.blogfa.com.
Uploaded: 2017-07-11T09:19:03+00:00
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Description: Downloaded from www.pharmacy123.blogfa.com کروماتوگرافی CHROMATOGRAPHY Downloaded from www.pharmacy123.blogfa.com.

Downloaded from www.pharmacy123.blogfa.com
کروماتوگرافی CHROMATOGRAPHY Downloaded from

What is chromatography?
The separation of a mixture of compounds based upon the differential partitioning of various analytes species between a mobile phase and a stationary phase. Downloaded from

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Chromatogram a plot of analyte signal as a function of elution time Downloaded from

Classification of Column Chromatographic Methods Downloaded from

The Effect of Migration Rates and Zone Broadening on Resolution Downloaded from

Migration Rates of Solutes :
The effectiveness of a chromatographic column in separating two solutes depends in part on the relative rates at which the two species are eluted . These rates in turn are determined by the ration of solute concentration in each of the two phases . Downloaded from

Distributive constants :
All chromatographic separations are based on differences in the extent to which solutes are distributed between the mobile and stationary phases . Downloaded from

Distribution Constants The distributive constant for a solute in chromatography is equal to the ratio of its molar concentration in the stationary phase to its molar concentration in the mobile phase . K = cs / cm where cs = molar concentration of the solute in the stationary phase and cm = molar concentration of the solute in the mobile phase Ideally , the distribution constant is constant over a wide range of solute concentration ; that is , Cs is directly proportional to Cm Downloaded from

Migration Rates of Solutes Retention Times The time it takes after sample injection for an analyte peak to reach the detector is called retention time. tR = L / vavg Where L = length of column vavg = average velocity of analyte Dead time Downloaded from

Retention time (tR) Retention time (tR) : The retention time is the time between injection of a sample and the appearance of a solute peak at the detector of a chromatographic column . Adjusted time (tR'): The time which the solute spend in stationary phase t’r : adjusted time t r : retention time tm : hold up time or dead time Downloaded from

Dead time or void time (tM / t0)
tM represent the time that the un retarded substance (mobile phase) spend in the column. calculation equation Downloaded from

Average linear velocity (µ)
µ is the average speed of mobile phase (Gas or liquid), through the column. µ is expressed by cm/sec or mL/min. Calculating equation: µ : is linear velocity of mobile phase L : is the column length tM : is the retention time of solute L tM cm /s L µ = µ = cm /s tM Downloaded from

Migration Rates of Solutes Velocity of Mobile Phase Velocity of mobile phase can be calculated using tM. u = L / tM Where L = length of column u = average velocity of an unretained species Dead time Downloaded from

Retention factor (K) K = tR - tM tM = The retention factor is an important experimental parameter that is widely used to compare the migration rates of solutes on column . K is the ratio of the amount of time that a solute spend in stationary and mobile phases. The retention factor is the rate at which a solute migrates through a column . K is calculating by equation below: t’R Downloaded from

Migration Rates of Solutes The Relationship Between Retention Time and Distribution Constant In order to relate the retention time of the solute to its distribution constant, we express its migration as a fraction of the velocity of the mobile phase. Vavg = u x fraction of time solute spends in mobile phase * Downloaded from * = capacity factor or retention factor

Migration Rates of Solutes Relative Migration Rates: Selectivity Factor The selectivity factor a for solutes A and B is defined as the ratio of the distribution constant of the more strongly retained solute ( B ) to the distribution constant for the less strongly held solute ( A ) . By this definition a is always greater than 1. a = KB/KA a = k’B / k’A a = [(tR)B - tM ] / [(tR)A – tM] The selectivity factor for two analytes in a column provides a measure of how well the column will separate the two . Downloaded from

Zone Broadening and Column Efficiency Methods for Describing Efficiency The theory is based on work by Martin and Synge in which they treated a chromatographic column as if it were a distillation column H = L/N H = theoretical plate height N= number of theoretical plates L= length of column The plate theory successfully accounts for the Gaussian shape and their rate of movement down a column, but fails to account for peak broadening in a mechanistic way. The rate theory was developed to make up for these shortcomings. Downloaded from

The Theory of Chromatography
Plate theory - older; developed by Martin & Synge Rate theory - currently in use today Downloaded from

Plate Theory - Martin & Synge 1954 Nobel Laureates
View column as divided into a number (N) of adjacent imaginary segments called theoretical plates within each theoretical plate complete equilibration of analytes between stationary and mobile phase occurs Downloaded from

Theoretical plate or column efficiency (N)
The plate theory needs to assume that the solute, during its passage through the column, is always in equilibrium with the mobile and stationary phases. But the equilibrium between the solute and phases never actually occurs. So to obtain this equilibrum, the column must divided in number of cell or plat. Every plat has a specific size and solute spend limite time in each plat. so in the existing of small plats, the solute will spend little time in each plat and it will elute fast. Downloaded from

Plate Theory - Martin & Synge 1954 Nobel Laureates
Significance? Greater separation occurs with: greater number of theoretical plates (N) as plate height (H or HETP) becomes smaller L = N H or H = L / N where L is length of column, N is number of plates, and H is height of plates Downloaded from

Methods for Describing Efficiency The theory is based on work by Martin and Synge in which they treated a chromatographic column as if it were a distillation column H = L/N H = (LW2)/(16tR2) N = 16 (tR/W)2 N = 5.54(tR/W1/2)2 Downloaded from

N can be Estimated Experimentally from a Chromatogram
N = 5.55 tr2 / w1/22 = 16 tr2 / w2 where: tr is retention time; w1/2 is full width at maximum w is width measured at baseline Downloaded from

Choice of Column Dimensions
Nmax = 0.4 * L/dp where: N - maximum column efficiency L - column length dp - particle size So, the smaller the particle size the higher the efficiency! Downloaded from

Efficiency Relative to Analysis Time
today 90 mm L 3 um today 150 mm L 5 um N 1970’s 300 mm L 10 um Analysis Time, min 10 Downloaded from 100

First Important Prediction of Plate Theory
Band spreading - the width of bands increases as their retention time (volume) increases . Downloaded from

Problem: A band exhibiting a width of 4 mL and a retention volume of 49 mL is eluted from a column. What width is expected for a band with a retention volume of 127 mL eluting from the same analyte mixture on the same column? ANS: mL Downloaded from

Second significant prediction of plate theory
The smaller HETP, the narrower the eluted peak Downloaded from

Plate Theory - Practical Considerations
Not unusual for a chromatography column to have millions of theoretical plates Columns often behave as if they have different numbers of plates for different solutes present in same mixture Downloaded from

Rate Theory Based on a random walk mechanism for the migration of molecules through a column takes into account: band broadening effect of rate of elution on band shape availability of different paths for different solute molecules to follow diffusion of solute along length Downloaded from

Van Deemter Equation H = A n1/3 + B/n + C n where: H is HETP (remember want a minimum!) n is mobile phase velocity A, B, and C are constants Downloaded from

Van Deemter Equation H = A n1/3 + B/n + C n first term - rate of mobile phase movement through column (often just a constant) second term - longitudinal solute diffusion; solute concentration always lower at edges of column so solute diffuses longitudinally third term - equilibration is not instantaneous Downloaded from

Zone Broadening and Column Efficiency Kinetic Variables Affecting Zone Broadening Downloaded from

Zone Broadening and Column Efficiency Kinetic Variables Affecting Zone Broadening Mobile-Phase Flow Rate Downloaded from

H = A + B/u + Cu H = A + B/u +(Cs + Cm)u
Zone Broadening and Column Efficiency Relationship Between Plate Height and Column Variables - van Deemter Equation H = A + B/u + Cu H = A + B/u +(Cs + Cm)u Downloaded from

Zone Broadening and Column Efficiency van Deemter Equation - The Multipath Term (A) H = A + B/u + Cu A = 2ldp l depends on particle size distribution, the narrower the distribution the smaller the l The smaller the particle size, the smaller the A term Independent of mobile phase flow rate Also known as eddy diffusion Downloaded from

Zone Broadening and Column Efficiency van Deemter Equation - Longitudinal Diffusion (B) Negative slope due to this term less for LC H = A + B/u + Cu B/u = 2gDM/u g is related to the diffusion restriction of packed columns, with packed columns this value is about 0.6 and 1 for open tubular columns DM is the mobile phase diffusion coefficient Inversely related to mobile phase flow rate Downloaded from

Zone Broadening and Column Efficiency van Deemter Equation - Mass Transfer (C) H = A + B/u +(Cs + Cm)u CS = fS(k’)df2 / DS CM = fM(k’)dp2 / DM f(x) is a function of x DM is the mobile phase diffusion coefficient DS is the stationary phase diffusion coefficient df is film thickness dp is particle size Directly related to mobile phase flow rate Downloaded from

Zone Broadening and Column Efficiency van Deemter Equation - Summary H = A + B/u +(Cs + Cm)u Downloaded from

Zone Broadening and Column Efficiency Effect of Particle Size on Plate Height Downloaded from

Optimization of Column Performance A chromatographic separation is optimized varying experimental conditions until the components of the mixture are separated cleanly with a minimum expenditure of time. Optimization experiments are aimed at reducing zone broadening altering relative migration rates of components Downloaded from

Resolution Ideal chromatogram exhibits a distinct separate peak for each solute Reality: chromatographic peaks often overlap We call the degree of separation of two peaks: Resolution = peak separation average peak width Downloaded from

Column resolution : The resolution of a chromatographic column is a quantitative measure of its ability to separate analytes A and B . Downloaded from

Resolution So, separation of mixtures depends on: width of solute peaks (want narrow) efficiency spacing between peaks (want large spacing) selectivity Downloaded from

Example What is the resolution of two Gaussian peaks of identical width (3.27 s) and height eluting at 67.3 s and 74.9 s, respectively? ANS: Resolution = 2.32 Downloaded from

Optimization of Column Performance Column Resolution Resolution (RS) of a column provides a quantitative measure of its ability to separate two analytes Downloaded from

Effect of Retention and Selectivity Factors on Resolution Column Resolution Resolution in terms of retention times and efficiency Resolution in terms of capacity factors and efficiency Downloaded from

Effect of Retention and Selectivity Factors on Resolution Column Resolution Resolution in terms of capacity factors, efficiency, and selectivity factors Efficiency in terms of capacity factors, resolution, and selectivity factors Downloaded from

Variables That Affect Column Performance Resolution in terms of capacity factors, efficiency, and selectivity factor First term related to the kinetics that lead to band broadening Second term is a selectivity term that is only related to the properties of the two solutes Third term depends on properties of both the solute and the column Downloaded from

Variation In Retention Factor Downloaded from

General Elution Problem Downloaded from

Important Chromatographic Terms Downloaded from

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